The overall aim is to contribute to the understanding of regulatory mechanisms operating in retrovirus infected cells using the expression of Rous sarcoma virus (RSV) genes as a model system. The particular emphasis of the project is on viral RNA processing and translation. Knowledge obtained by studying this system should be applicable to similar problems in pathogenic human retroviruses such as HTLV III/LAV as well as to our understanding of viral oncogenesis. Furthermore, the results obtained in the studies should be applicable to other eukaryotic systems where alternate RNA splicing and translational controls play important regulatory roles. Two aspects of regulation will be investigated: 1) Cis-acting sequences and trans-acting viral and cellular factors required to regulate the intracellular pools of spliced and unspliced virus-specific RNA will be studied. Deletion and site-specific mutagenesis of infectious plasmids will be used to study cis-acting elements. Transient expression assays for splicing of viral RNA in the presence of viral gag gene proteins will be carried out to investigate the role of virus-specific transacting factors. Mammalian-avian cell fusions and in vitro splicing assays will be used to determine the effects of cellular trans-acting factors on splicing. The potential role of N-6-methyladenosine (m6A) methylations in splicing of viral RNA will be studies by the analysis of intracellular viral RNA precursors for methylations any by site-specific mutagenesis of methylation sites in the viral genome. 2) The role of the unique mRNA 5' leader sequence and upstream AUGs in modulating the translation of pp60src will be investigated. The effects of deleting the upstream open reading frames, increasing the length of the upstream open reading frames, and substituting sequences predicted to promote ribosome "fall-off" between the termination codon of the upstream open reading frame and downstream srg gene AUG will be determined. The experiments will be carried out in vivo by transfection of appropriate src gene cDNA clones and in vitro, by translation of SP6 phage polymerase-generated RNA obtained by transcription of src gene cDNA clones. The role of trans-acting viral and cellular factors that interact with the 5' leader and promote translation will also be investigated. Expression of src cDNA clones transfected into transformation-defective RSV-infected and uninfected cells will be compared. Transfections will also be carried out in the presence of individual viral gag proteins.